Spectrophotometer for measurement of derivative spectra

ABSTRACT

A spectrophotometer for measurement of the derivative spectra of substances, comprising a monochromatic light source having a spectral bandwidth, a beam divider for dividing the light from the light source into two beams, masks separate from and positioned optically downstream from the beam divider for partially masking the beams so that the unmasked portion of each of the beams has a nominal wavelength different by a predetermined value from that of the unmasked portion of the other beam, a sample container so arranged as to receive the unmasked portions of the light beams and a detector to measure the intensities of light beams transmitted through the sample cells.

ited States Patent 1191 1111 3,737,234

Shiloata et a1. 1 51 June 5, 1973 1541 SPECTROPHOTOMETER FOR 2,547,212 41951 Jamison et al. ..2s0/43.5 R MEASUREMENT OF DERIVATIVE 2,650,3078/1953 Koppius 250 435 R 3,520,614 7/1970 Goldstein ..356/95 X 3,547,54212 1970 Bulpitt et al. ..356/88 x Inventors: Kazuo Shibata, Tokyo;Takashi Kurita, Kyoto, both of J apan Assignee: Shimadzu SeisakushoLtd., Kyoto,

Japan Filed; Dec. 17, 1970 Appl. No.: 99,140

Related U.S. Application Data Continuation-impart of Ser. No. 571,458,Aug. 10, 1966, abandoned.

U.S. Cl. ..356/88, 356/95, 356/97 Int. Cl ..G01j 3/42 Field of Search..250/43.5 R;

References Cited UNITED STATES PATENTS 10/1965 Frei et a1. ..356/97Primary Examiner-Ronald L. Wibert Assistant ExaminerF. L. EvansAttorney-Fidelman, Wolffe & Leitner [57] ABSTRACT A spectrophotometerfor measurement of the derivative spectra of substances, comprising amonochromatic light source having a spectral bandwidth, a beam dividerfor dividing the light from the light source into two beams, masksseparate from and positioned optically downstream from the beam dividerfor partially masking the beams so that the unmasked portion of each ofthe beams has a nominal wavelength different by a predetermined valuefrom that of the unmasked portion of the other beam, a sample containerso arranged as to receive the unmasked portions of the light beams and adetector to measure the intensities of light beams transmitted throughthe sample cells.

5 Claims, 9 Drawing Figures RATIO OF LOG 0F \SCANNER 28/ 27 RATIOPATENTEDJUH slam 3.737.234

SHEET 2 OF 3 RATIO OF SIGNALS LOG 0F \SCANNER 28 27 RATIO FIG. 4

WAVELENGT H M FIG. 6

INTENSITY II WAVELENGTH WAVELENGTH SPECTROPHOTOMETER FOR MEASUREMENT OFDERIVATIVE SPECTRA This invention is a continuation-in-part applicationof earlier-filed pending application Ser. No. 571,458, filed Aug. 10,1966 now abandoned.

This invention relates generally to absorption spectrophotometry andmore particularly to a spectrophotometer which is capable of measuringthe derivative spectra of various substances.

It is well known that an absorption spectrum of a substance, especiallya biological one, obtained by the usual method of spectrophotometricmeasurement often shows a single absorption band where there shouldactually appear a plurality of overlapping absorption bands, or anobscure shoulder where there is a weak absorption band superimposed onthe steep slope of a sharp absorption band. The corresponding derivativespectrum, however, can resolve such an apparently single band into itsactual components, or show such a shoulder as a distinct absorptionband.

One prior art instrument for measurement of derivative spectra employs apair of monochromators each adapted to provide a monochromatic light ofa wavelength slightly different from that provided by the othermonochromator. The two monochromatic light beams are passed through thesame sample contained in a pair of cells, and by measuring the ratio ofthe intensitites of light transmitted through the respective cells, thederivative spectrum of the sample is obtained. This instrument has thedisadvantage that it requires two monochromators, and that it is verydifficult to carry out scanning with the same difference being alwayskept between the wavelengths of the light beams of the twomonochromators.

Another prior art instrument employs a vibrating slit mechanism toprovide alternating monochromatic light beams of slightly differentwavelengths, together with two servo systems for compensating forvariation of the sensitivity of the photoelectric tubes and the lightintensity with wavelength, and complicated electric circuits forrecording derivative spectra. The disadvantage here is that thearrangements are rather complicated.

Accordingly, it is one object of the invention to provide aspectrophotometer which is capable of measuring the first derivativespectra of various substances.

Another object of the invention is to provide such a spectrophotometeras aforesaid which is very simple in construction, employing a singledouble-beam monochromator.

Another object of the invention is to, provide such a spectrophotometeras aforesaid which is easy for manufacture with a high degree ofprecision.

A further object of the invention is to provide such a spectrophotometeras aforesaid, utilizing the system of any conventional double-beamspectrophotometer.

Still another object of the invention is to make it possible to convertany conventional double-beam type of spectrophotometer into the onewhich is capable of measuring the derivative spectra of substances,without any modification of the basic arrangement of the instrument, butwith addition of only two simple masks.

Other objects, features and advantages of the invention will become moreapparent from the following course of specification, reference being hadto the accompanying drawings, wherein:

FIG. 1 is a graph showing a hypothetical absorption spectrum obtained bythe usual method of measurement;

FIG. 2 shows the derivative spectrum of a sample having the hypotheticalabsorption spectrum of FIG. 1;

FIG. 3 is a graph showing a hypothetical absorption spectrum obtained bythe ordinary method and the derivative thereof, for illustration of howan obscure shoulder in the ordinary absorption curve appears as adistinct absorption band in the derivative curve;

FIG. 4 is a schematic showing of one embodiment of the invention;

FIG. 5 is a schematic elevational view of the pair of masks with theirrespective slits shown in FIG. 4;

FIG. 5a is a view similar to FIG. 5 showing an arrangement to vary thewidth of the slits of the masks;

FIG. 6 shows graphs illustrating how the two monochromatic light beamsat slightly different wavelengths are partially and symmetricallyintercepted by the masks of the invention; and

FIG. 7 is a diagram of another embodiment of the invention; and

FIG. 8 is a diagram of an electrical circuit which can replace thecorresponding circuits in FIGS. 4 and 7.

Now referring in detail to the drawings, first to FIG. 1, there is showna hypothetical absorption curve of a sample plotted against wavelength.Let the absorbances at wavelengths of A, and A be A, and A respectively,and the following equations will be obtained:

where I, is the intensity of light incident on the sample at awavelength of A I is the intensity oflight incident on the sample at awavelength of i I, is the intensity of light transmitted through thesample at the wavelength of A and I, is the intensity of lighttransmitted through the sample at the wavelength of A IfA A A A, then AAlog 02/ 0) g ot/ n) Under the condition that the intensities of the twoincident light beams 1,, and I are equal, the equation (3) can beexpressed as:

A A g( n/ 2) 4 If both sides of this equation (4) are divided by AA,then A AM A g n/ 12) (5) On the other hand, the differential value ofthe absorbance A of the sample with respect to wavelength A is given as:11

dA/dh AA 0 AA/AA 6 If in. equation (5) the value AA is kept constant atnearly zero, the value of AA/AA can be considered as being approximatelyequal to dA/dk. This means that measurement of AA/AA with continuouswavelength scanning will approximately give the derivative spectrum ofthe sample.

As previously mentioned, derivative spectra are useful in detectingoverlapping absorption bands appearing as a single band or a distinctabsorption band appearing as an obscure shoulder in a usual absorptionspectrum. As shown in FIG. 3, a usual absorption curve is plotted withan obscure shoulder S, which clearly appears as a deep dip P in thederivative curve A.

According to the present invention there is provided a spectrophotometerfor measurement of derivative spectra of substances, which comprises amonochoram tic light source having a certain spectral bandwidth and abeam divider for dividing the light from the light source into twobeams. The characteristic of the invention is that to obtain thederivative of spectra of substances to be measured, there are provided apair of masks separate from and positioned optically downstream from thebeam divider for partially masking the two beams so that the unmaskedportion of each of the beams has a wavelength different by apredetermined value from that of the unmasked portion of the other beam.In one embodiment of the invention, the unmasked portions of the twobeams are passed through two sample cells containing the same sample,respectively, and the light beams transmitted through the sample cellsare received by two detectors, which produce electrical signalscorresponding to the intensities of the transmitted light beams. Inanother embodiment of the invention, the unmasked portions of the twobeams are alternately passed through the single sample cell and thelight beams transmitted through the single sample cell are received by asingle detector, which produces electrical signals corresponding to theintensities of the transmitted light beams.

The electrical signals are processed by means of a suitable electricalcircuit to provide a logarithmic ratio of the signals, which is plottedagainst wavelength to provide a derivative spectrum of the substancebeing measured.

Turning now to FIG. 4 which shows one embodiment of the invention, thereis shown a light source 10. The light from the source is reflected by aconcave mirror 11 and a pair of planemirrors l2 and 13 to enter amonochromator 14 through an entrance slit 15 and hit on a collimatormirror 16. The collimator mirror 16 makes the light rays parallel anddirects them to a prism 17, which is driven by a wavelength (orwavenumber) scanning device 31in a manner well known in the art. Theprism disperses the light rays into different wavelengths, which aredirected back to the collimator mirror l6 and again passed through theslit 15. The slit passes a monochromatic light of a selected spectralbandwidth, which passes through a collimator lens 30 and a chopper 18 toa beam splitter 19. The beam splitter splits the monochromatic lightrays into two beams B and B, which are reflected by concave mirrors 20and 20' to pass through optical attenuators 21 and 21, cylindricallenses 22 and 22' and sample cells C and C, respectively. Formeasurement of a derivative spectrum, the two cells contain the samesample. The light beams transmitted through the cells are received by apair of photomultiplier tubes 23 and 23, which produce correspondingoutputs.

The outputs from the tubes 23 and 23' may be applied to a suitablecircuit 24 which is so designed as to provide a signal corresponding tothe ratio .of the two signals from the tubes. The output from'thecircuit 24 is applied to a circuit 25 which is so designed as to providea signal corresponding to the logarithm of the ratio. The output fromthe circuit 25 is applied to a recorder 32. If the chart 29 of therecorder is moved in accordance with the wavelength or wavenumber scan-4 ning by the device 31, a pen 27 draws a derivative spectrum 28 on thechart 29.

Characteristic of the invention is the provision of a pair of masks Mand M for obtaining beams of slightly different wavelengths in the twopaths of the beams B and B in front of the cells C and C, respectively.The masks M and M are positioned in the image plane of the slit 15 ofthe monochromator and formed with slits S and S, respectively. The slitsare of the same width and, as most clearly shown in FIG. 5, so formedthat the position of the slit S relative to the optical axis of the beamB is displaced from that of the slit S relative to the optical axis ofthe beam B. In the illustrated embodiment, the slit S on the mask M isdisplaced slightly (for example, 0.5 mg around A 400 my.) toward thelonger wavelengths while the slit S on the mask M is displacedsymmetrically toward the shorter wavelengths. Thus, the two beams B andB have their respective symmetrical portions intercepted by the masks Mand M. Consequently, although the two beams that have been split fromthe same single monochromatic beam have the same small spectralbandwidth, there comes to exist a slight difference AA between the twonominal wavelengths A and A, of the beams that have passed through theslits S and S. Then the sample in the cell C receives the light from theslit S, the nominal wavelength of which is A and the intensity of whichis l while the sample in the cell C receives the light from the slit S,the nominal wavelength of which is A and the intensity of which is 1 Thelight beams transmitted through the sample in the cells C and C arereduced to the intensities of 1, and 1 respectively, which are detectedby the photomultiplier tubes 23 and 23. The output signals of the tubesare amplified and demodulated and then measured as the ratio of I /1,the logarithm of which gives the absorbance difference between thetwowavelengths A and A Thus, the derivative spectrum of the sample canbe continuously recorded against wavelength A, as previously mentioned.

It will be easily understood that by varying the width w of the slits Sand S and/or the central distance D (FIG. 5) between the slits withinthe spectral bandwidth of the beams hitting the masks, it is possible tovary the value of AA. The width w of the slits S, S may be varied byproviding a pair of plates m, n and m, n, which are slidably secured tothe masks M,M' so as to be movable toward and away from each other infront of the slits 8,8. I

FIG. 6 shows the principle of the simplest method of masking, that is,one-half masking of the light beams B and B incident on the sample cellsC and C. Suppose that the beams B and B have the spectral intensitydistribution as plotted against wavelength in FIG. 6a. Onehalf of one ofthe beams is masked so that the nominal wavelength .of the unmaskedportion'(shown hatched in the Figures) of the beam is slightly shiftedsay, to A as shown in FIG. 6b. The opposite one-half of the other beamis shifted to A as shown in FIG. 60. Thus, a difference AA A A, isproduced between the two light beams incident on the sample cells C andC.

FIG. 7 shows another embodiment of the invention,

1 wherein a single cell and a single photomultiplier tube are employedinstead of the two cells C, C' and the two photomultiplier tubes 23, 23'in FIG. 4. In FIG. 7, the same reference symbols and numerals as in FIG.4 denote corresponding parts. The beam coming out of the slit 15 isdirected by a beam switch 34a alternately onto the concave mirrors and20. The two alternate beams B and B pass through the attenuators 21 and21, the lenses 22 and 22 and the masks M and M and are reflected by apair of mirrors 33 and 33' to be alternately projected onto a secondbeam switch 34b. The two beam switches 34a and 34b are synchronouslydriven by a suitable drive 35. The switch 34b causes the two beams toalternately pass through a single cell 36 into a single photomultipliertube 37. The alternate outputs corresponding to the absorption of thesample at the two slightly different wavelengths are demodulated by ademodulator 38 which is synchronized with the operation of switches 34aand 34b. The outputs from the demodulator 38 are applied to smoothingcircuits 39 and 39, the outputs from which are applied to the ratiocalculating circuit 24. The other component parts and the operation ofthe system of FIG. 7 are the same as in FIG. 4 so that no furtherexplanation will be required.

In FIGS. 4 and 7, first the ratio of the two outputs from thetubes 23and 23 or the smoothing circuits 39 and 39 is calculated and then thelogarithm of the ratio is provided. However, the arrangement may also besuch that a signal corresponding to the logarithm of each of the twooutputs is first obtained and then the difference between the twologarithms is calculated. FIG. 8 shows such an arrangement. The outputsfrom the tubes 23 and 23 in FIG. 4 or the smoothing circuits 39 and 39'in FIG. 7 are applied to a pair of circuits 40 I and 40', respectively,which are so designed as to produce a signal corresponding to thelogarithm of the input signal thereto. The outputs from the circuits 40and 40 are applied to a circuit 41 which is so designed as to produce anoutput signal corresponding to the difference between the two outputslogarithms from the circuits 40 and 40. The output signal from thecircuit 41 is applied to the recorder 32 in FIG. 4 or 7.

In FIG. 7, the beam switch 34a alternately directs the light from themonochromator to advance along the two paths and the second beam switch34b causes the two beams to be projected through the single sample cell.The beam switch 34a may be replaced by a beam splitter similar to theone shown in FIG. 4 and the second beam switch 34b, by a device whichcauses the two beams B and B on the two paths to be projected throughthe single cell, with a device disposed in the two beams so as toalternately intercept them so that they alternately pass through thesingle cell.

Having illustrated and described a preferred embodiment of theinvention, it is to be understood that the invention is not limitedthereto, and that there are many modifications and changes thereofwithout departing from the true scope and spirit of the invention asdefined in the appended claims.

What is claimed is:

l. A spectrophotometer for measurement of derivative spectra ofsubstances, comprising: means for providing monochromatic light, meansfor dividing said monochromatic light into two separate beams having thesame spectral bandwidth; means separate from and positioned opticallydownstream from said dividing means in each of said beams for partiallymasking each said beam so that the unmasked portion of each said beamhas a wavelength different by a predetermined value from that of theunmasked portion of the other beam; said masking means comprises a pairof plates each having a slit formed therein, the position of the slit onone of said plates relative to the optical axis of one of said beamsbeing symmetrically displaced from the position of the slit on the otherof said plates relative to the optical axis of the other of said beams;a pair of cells, one in each of said beams for containing a portion ofthe same sample to receive said unmasked portions of the light beams,respectively; a pair of means for detecting the intensities of lightbeams transmitted through said samples to generate electrical signalscorresponding to said intensities of transmitted light; means formeasuring said electrical signals to provide the ratio ofwsaidintensities of transmitted light; means for providing the logarithm ofsaid ratio; and means for plotting the same against wavelength toprovide a derivative spectra output record.

2. The spectrophotometer as defined in claim 1, wherein said first-namedmeans comprises a single monochromator.

3. The spectrophotometer as defined in claim 1, wherein the widths ofbothsaid slits are variable.

4. The spectrophotometer as defined in claim 1, wherein saiddisplacement of each of said slits is variable.

5. A spectrophotometer for measurement of deriva tive spectra ofsubstances comprising: means for providing monochromatic light, meansfor dividing said monochromatic light into two separate beams having thesame spectral bandwidth; means separate from and positioned opticallydownstream from said dividing means in each of said beams for partiallymasking each said beam so that the unmasked portion of each said beamhas a wavelength different by a predetermined value from that of theunmasked portion of the other beam; said masking means comprising a pairof plates each having a slit formed therein, the positioned of the sliton one of said plates relative to the optical axis of one of said beamsbeing symmetrically displaced from the position of the slit on the otherof said plates relative to the optical axis of the other of said beams;a pair of cells, one in each of said beams for containing a portion ofthe same sample, to receive said unmasked portions of the light beams,respectively; a pair of means for detecting the intensities of lightbeams transmitted through said sample cells to generate electricalsignals corresponding to'said intensities of transmitted light; meansfor providing the logarithms of each of said electrical signals; meansfor providing an electrical signal corresponding to the differencebetween said logarithms; and means for recording said last-mentionedelectrical signal to provide a derivative spectra output record.

1. A spectrophotometer for measurement of derivative spectra ofsubstances, comprising: means for providing monochromatic light, meansfor dividing said monochromatic light into two separate beams having thesame spectral bandwidth; means separate from and positioned opticallydownstream from said dividing means in each of said beams for partiallymasking each said beam so that the unmasked portion of each said beamhas a wavelength different by a predetermined value from that of theunmasked portion of the other beam; said masking means comprises a pairof plates each having a slit formed therein, the position of the slit onone of said plates relative to the optical axis of one of said beamsbeing symmetrically displaced from the position of the slit on the otherof said plates relative to the optical axis of the other of said beams;a pair of cells, one in each of said beams for containing a portion ofthe same sample to receive said unmasked portions of the light beams,respectively; a pair of means for detecting the intensities of lightbeams transmitted through said samples to generate electrical signalscorresponding to said intensities of transmitted light; means formeasuring said electrical signals to provide the ratio of saidintensities of transmitted light; means for providing the logarithm ofsaid ratio; and means for plotting tHe same against wavelength toprovide a derivative spectra output record.
 2. The spectrophotometer asdefined in claim 1, wherein said first-named means comprises a singlemonochromator.
 3. The spectrophotometer as defined in claim 1, whereinthe widths of both said slits are variable.
 4. The spectrophotometer asdefined in claim 1, wherein said displacement of each of said slits isvariable.
 5. A spectrophotometer for measurement of derivative spectraof substances comprising: means for providing monochromatic light, meansfor dividing said monochromatic light into two separate beams having thesame spectral bandwidth; means separate from and positioned opticallydownstream from said dividing means in each of said beams for partiallymasking each said beam so that the unmasked portion of each said beamhas a wavelength different by a predetermined value from that of theunmasked portion of the other beam; said masking means comprising a pairof plates each having a slit formed therein, the positioned of the sliton one of said plates relative to the optical axis of one of said beamsbeing symmetrically displaced from the position of the slit on the otherof said plates relative to the optical axis of the other of said beams;a pair of cells, one in each of said beams for containing a portion ofthe same sample, to receive said unmasked portions of the light beams,respectively; a pair of means for detecting the intensities of lightbeams transmitted through said sample cells to generate electricalsignals corresponding to said intensities of transmitted light; meansfor providing the logarithms of each of said electrical signals; meansfor providing an electrical signal corresponding to the differencebetween said logarithms; and means for recording said last-mentionedelectrical signal to provide a derivative spectra output record.